Oncogene (2014) 33, 3441–3450 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc ORIGINAL ARTICLE Identification of RBCK1 as a novel regulator of FKBPL: implications for tumor growth and response to tamoxifen C Donley1,4, K McClelland1,4, HD McKeen1,4, L Nelson1, A Yakkundi1, PV Jithesh2, J Burrows1, L McClements1, A Valentine1, KM Prise3, HO McCarthy1 and T Robson1 FKBPL has been implicated in processes associated with cancer, including regulation of tumor growth and angiogenesis with high levels of FKBPL prognosticating for improved patient survival. Understanding how FKBPL levels are controlled within the cell is therefore critical. We have identified a novel role for RBCK1 as an FKBPL-interacting protein, which regulates FKBPL stability at the post-translational level via ubiquitination. Both RBCK1 and FKBPL are upregulated by 17-b-estradiol and interact within heat shock protein 90 chaperone complexes, together with estrogen receptor-a (ERa). Furthermore, FKBPL and RBCK1 associate with ERa at the promoter of the estrogen responsive gene, pS2, and regulate pS2 levels. MCF-7 clones stably overexpressing RBCK1 were shown to have reduced proliferation and increased levels of FKBPL and p21. Furthermore, these clones were resistant to tamoxifen therapy, suggesting that RBCK1 could be a predictive marker of response to endocrine therapy. RBCK1 knockdown using targeted small interfering RNA resulted in increased proliferation and increased sensitivity to tamoxifen treatment. Moreover, in support of our in vitro data, analysis of mRNA microarray data sets demonstrated that high levels of FKBPL and RBCK1 correlated with increased patient survival, whereas high RBCK1 predicted for a poor response to tamoxifen. Our findings support a role for RBCK1 in the regulation of FKBPL with important implications for estrogen receptor signaling, cell proliferation and response to endocrine therapy. Oncogene (2014) 33, 3441–3450; doi:10.1038/onc.2013.306; published online 5 August 2013 Keywords: RBCK1; FKBPL; estrogen receptor; p21; tamoxifen INTRODUCTION these data support a role for FKBPL as a tumor suppressor and, The FKBPL gene encodes a protein with a molecular mass of therefore, understanding how this protein is regulated is critical. B39 kDa, with two characteristic regions, the tetratricopeptide RBCK1 (HOIL-1) encodes a 510 amino-acid protein with several repeat domain and a partial peptidyl-prolyl-cis-trans-isomerase distinct functional domains: a RING-IBR domain, B-Box, two coiled- 11 domain. These domains are features of the FKBP/immunophilin coil regions and a ubiquitin-like sequence. The RING-IBR consists family of which FKBPL represents a divergent member. The of two RING fingers and an in-between RING-finger motif making tetratricopeptide repeat domain is required for protein–protein it homologous with E3 ubiquitin ligase enzymes. Functional interactions1 and facilitates interaction with the cellular analysis has identified RBCK1 as an E3 ligase involved in the chaperone, heat shock protein (Hsp)90.2 Through its interaction ubiquitination of PKC, IRP2, SOCS6, BACH1, TAB2/3 and IRF3 12–16 with Hsp90, FKBPL has been shown to regulate the stability proteins. RBCK1 is also involved in the 600-kDa heteromeric of the cyclin-dependent kinase inhibitor, p213,4 and also form linear ubiquitin chain assembly complex17,18 responsible for the co-chaperone complexes with the glucocorticoid receptor, linear polyubiquitination of NF-kappa-B essential modulator androgen receptor and estrogen receptor-a (ERa).5–7 FKBPL (NEMO) resulting in activation of the nuclear factor-kB pathway directly regulates protein levels of ERa and its phosphorylation involved in immune and inflammatory responses.19,20 RBCK1 has at Ser118, sensitizing ER-positive breast cancer cell lines to been implicated in modulating cell growth through regulation of estrogen deprivation and endocrine therapy.6 Overexpression of p53 activation of p21 transcription.21 RBCK1 contains DNA-binding FKBPL inhibited the growth of several tumor cell lines,6,8 and high domains and directly influences the transactivation of numerous FKBPL was also found to correlate with both increased overall genes including, p21, HBv-X and Eya1.21–23 It also positively survival and distant metastasis-free survival in microarray data regulates ERa transcription by enhancing promoter binding, sets.6 This was further supported in a breast cancer tissue resulting in transactivation of ER responsive genes, c-myc, Cyclin microarray where high FKBPL was associated with increased D and pS2.24,25 Here, we show that RBCK1 is a post-transcriptional survival and reduced tumor size and grade,9 highlighting its regulator of FKBPL, stabilizing FKBPL through ubiquitination. prognostic value. FKBPL’s prognostic potential may also be related We further show that RBCK1 acts as a co-chaperone within to its anti-angiogenic activity; secreted FKBPL and a therapeutic Hsp90/FKBPL/ERa complexes, regulating a number of processes, peptide derivative inhibit angiogenesis in vitro and in vivo, result- including ERa transactivation in association with FKBPL, response ing in inhibition of human tumor xenograft growth.10 Together, to endocrine therapy and cell proliferation. 1School of Pharmacy, McClay Research Centre, Queen’s University, Belfast, Northern Ireland; 2Liverpool Cancer Research UK Centre, University of Liverpool, Liverpool, UK and 3Centre for Cancer Research and Cell Biology, Queen’s University, Belfast, Northern Ireland. Correspondence: Professor T Robson, School of Pharmacy, McClay Research Centre, Queen’s University, 97 Lisburn Road, Belfast, Country Antrim BT9 7BL, Northern Ireland. E-mail: [email protected] 4These authors contributed equally to this work. Received 21 January 2013; revised 27 May 2013; accepted 11 June 2013; published online 5 August 2013 RBCK1 modulates FKBPL and affects tumor growth C Donley et al 3442 RESULTS a characteristic ubiquitin smear when co-transfected with either RBCK1 is a novel FKBPL-interacting protein involved in FKBPL full-length or RBCK1DRING mutant; no smear was observed when ubiquitination transfected with the RBCK1DUbl mutant (Figure 1e). This suggests An yeast-2-hybrid screen was used to isolate FKBPL-interacting that the Ubl domain of RBCK1 is required for the ubiquitination of proteins, identified by their ability to induce expression of three FKBPL. To assess the type of ubiquitin chain that RBCK1 attaches independent reporter genes enabling yeast cell growth on amino- to FKBPL, two ubiquitin mutants were utilized, one with all lysine acid drop out media, inducing b-gal activity and inhibiting yeast residues mutated to arginine, except K48, and the other with only growth on media containing 5-fluoroorotic acid (Figure 1a). Three K48 mutated to arginine (Supplementary Figure 1c); wild-type independent clones of RBCK1 were identified as strong interacting ubiquitin was used as a control. FKBPL was ubiquitinated in the partners; these clones spanned the Ubl and RING domains of presence of both mutants and wild-type ubiquitin (Figure 1f), RBCK1. The mammalian two-hybrid assay was used to confirm and suggesting that the ubiquitin chains attached to FKBPL are not map the interacting domains. Co-transfection of full-length FKBPL linked via any of the seven classical lysine residues. RBCK1 has and RBCK1 resulted in a 20-fold induction in firefly luminescence been shown to work in tandem with RNF3 to form linear ubiquitin over the self-activation control, indicating a strong interaction chains that are formed by N to C terminal conjugation rather than 14,16,17,19 (Figure 1b). Deletion of the tetratricopeptide repeat domain in from lysine residues. RBCK1 could therefore modify FKBPL FKBPLD200, FKBPLD151 or FKBPLD86 constructs (Supplementary via this type of ubiquitination. Figure 1a) abrogated the interaction with RBCK1. The interaction was confirmed using pull-down assays; full-length but not trun- RBCK1 regulates FKBPL protein levels cated FKBPLD200 interacted with myc-tagged RBCK1 (Figure 1c). To assess the functional potential of the interaction between Mutational analysis of RBCK1 demonstrated an interaction bet- RBCK1 and FKBPL, RBCK1 was transiently overexpressed in MCF-7 ween FKBPL and the myc-tagged RBCK1/DRING mutant, but not cells at both the mRNA and protein levels (Figure 2a). This the RBCK1/DUbl mutant, confirming that the Ubl was important coincided with a significant increase in FKBPL protein (Po0.05), for its interaction with FKBPL, although the RING domain may still whereas FKBPL mRNA levels remained constant (Figure 2a and be needed to enhance this interaction (Figure 1d and Supplemen- Supplementary Figure 2a). MCF-7 clones stably overexpressing tary Figure 1b). To test whether RBCK1 could alter the ubiquitin RBCK1, 3.1 Â 8 and 3.1 Â 10, had increased levels of RBCK1 at status of FKBPL, we assessed the ability of full-length RBCK1 and the mRNA and protein levels (Figure 2b and Supplementary mutated RBCK1 (lacking either the Ubl or RING domain) to Figure 2b). In support of the transient transfections, FKBPL protein ubiquitinate FKBPL. Immunoprecipitation of FKBPL resulted in levels were significantly elevated in both RBCK1 clones compared Figure 1. Full-length FKBPL interacts with RBCK1. (A) A yeast two-hybrid positive clone obtained from screening a human fetal brain library with full-length FKBPL. The detection of (a) b-galactosidase activity, (b, c) yeast colony growth on drop-out media (SC-Leu-Trp-His þ 10 mM 3AT or SC-Leu-Trp-Ura) and (d) yeast growth inhibition on SC-Leu-Trp þ 5FOA represented a positive protein interaction. (B) Mammalian two-hybrid assay demonstrating interaction between RBCK1 and full-length FKBPL but not FKBPL-truncated mutants in L132 cells (n ¼ 4). (C) Co-immunoprecipitation in COS-7 lysates immunoabsorbed to protein G-Sepharose beads with antibodies against MYC-tagged RBCK1 and HA-tagged FKBPL. (D) Co-immunoprecipitation in COS-7 lysates immunoabsorbed to protein G-Sepharose beads with antibodies against MYC-tagged RBCK1 and FKBPL. (E) T47D cells transfected with myc-tagged RBCK1 (full-length RBCK1 or truncated mutants), immunoprecipitated with FKBPL antibody and probed for HA-ubiquitin.